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Background and Objectives: Pooled nucleic acid amplification techniques (NAT) and donor screening for anti-hepatitis C virus (HCV) have reduced the diagnostic window period of HCV infection in the blood donor population from about 12 to 1 or 2 weeks. During that time, HCV RNA is hardly detectable by pooled or individual donation NAT. Here we describe a case of transfusion-acquired HCV infection from an extremely low-titre donation. After a repeat donor tested positive for HCV, a look-back procedure was initiated. A recipient of a red cell concentrate from the previous donation was identified and found to be infected with HCV as well. We compared several commercial NAT systems for their ability to detect the viraemic plasma., Materials and Methods: Molecular analyses of HCV in donor and recipient samples were performed. The HCV-transmitting plasma was tested using different commercially available qualitative and quantitative NAT assays., Results: HCV transmission was verified by molecular analyses and was assigned to genotype 2b. NAT with various commercial HCV assays detected the infection erratically in individual donations. However, the detection rate was not directly related to the claimed sensitivity of some HCV NATs., Conclusions: HCV transmission can be caused by donations that escape NAT detection even when tested in an individual donation. Comparison of different assays led to results that did not necessarily reflect the expected sensitivities. The need for standard materials representing further HCV genotypes is discussed.

Objective: Logistics and cost of nucleic acid amplification testing (NAT) screening preclude its current use in many developing countries. Development of hepatitis C virus (HCV) core antigen assays offer an alternative to NAT. We evaluated two specimen populations to assess the sensitivity, relative to NAT, of the HCV core antigen (HCVcAg) ELISA (enzyme-linked immunosorbent assay) test system and the trak-C assay: (1) plasma donor HCV NAT-conversion panels and (2) cross-sectional whole blood donor NAT yield specimens., Methods: Differential sensitivities among NAT (NGI; Chiron/Gen-Probe) and both HCVcAg assays (Ortho-Clinical Diagnostics, Rochester, NY) were evaluated using: (1) 102 serial ramp-up phase specimens from 37 plasma donor NAT-conversion panels (Alpha Therapeutic/BioClinical Partners); and (2) 42 cross-sectional whole blood donor NAT yield specimens (confirmed RNA positive, antibody negative) plus 54 NAT false-positive specimens (American Red Cross)., Results: Viral load among the plasma donor NAT-conversion panels at the cutoffs for HCVcAg and trak-C assays were 32 000 copies/ml (95% confidence interval [CI] 8000-120 000) and 8000 copies/ml (95% CI: 2200-28 000), respectively. The mean (95% CI) difference in window period reduction compared to routine mini-pool NAT screening (estimated sensitivity 100 copies/ml) was delayed 5.2 days (2.2-7.6 days) for HCVcAg assay and 3.8 days (2.1-5.5 days) for the trak-C assay. Among the 42 NAT yield specimens, the HCVcAg assay detected 31 (74%) as core antigen-positive while the trak-C assay detected 37 (88%) as core antigen-positive. Viral loads for the five specimens not detected by the trak-C HCVcAg assay ranged from 100 to 7770 copies/ml. All 54 NAT false-positive specimens were non-reactive on both HCV core antigen assays., Conclusion: These data indicate that the trak-C assay has sensitivity approaching routine mini-pool NAT screening for the detection of seronegative HCV infection. In the absence of routine NAT screening for early HCV infection, the use of an HCV core antigen assay should be considered.

Background and Objectives: A collaborative study was undertaken to establish a replacement for the current (1st) World Health Organization (WHO) hepatitis C virus (HCV) International Standard, 96/790., Materials and Methods: Both the 1(st) International Standard and the replacement standard were prepared from the same starting material by diluting a high titre genotype 1a HCV isolate in pooled, human plasma. The only difference was that each standard was lyophilized in two, separate lyophilisation runs but under the same conditions., Results: In the study to establish the 1st International Standard, no significant difference in potency was found between the material eventually designated as the 1st International Standard and that now selected as the 2nd International Standard. The present study also showed no significant differences between the materials stored at -20 degrees C and no evidence of degradation over 5 years., Conclusions: Material 96/798 was established as the 2nd HCV International Standard and assigned the same unitage as the 1st International Standard, i.e. 10(5) IU/ml (50,000 IU/vial).

Hepatitis B virus (HBV) presents a higher residual risk of transmission by transfusion than hepatitis C virus (HCV) or human immunodeficiency virus (HIV). While most infectious blood units are removed by screening for hepatitis B surface antigen (HBsAg), there is clear evidence that transmission by HBsAg-negative components occurs, in part, during the serologically negative window period, but more so during the late stages of infection. Donations negative for HBsAg, but positive for HBV DNA, with or without the presence of HBV antibodies, correspond to 'occult' HBV infection (OBI). The frequency of OBI depends on the relative sensitivity of both HBsAg and HBV DNA assays. It also depends on the prevalence of HBV infection in the population. OBI may follow recovery from infection, displaying antibody to hepatitis B surface antigen (anti-HBs) and persistent low-level viraemia, escape mutants undetected by the HBsAg assays, or healthy carriage with antibodies to hepatitis B e antigen (anti-HBe) and to hepatitis B core antigen (anti-HBc). Over time, in the latter situation, anti-HBe and, later, anti-HBc may become undetectable. The critical question is whether or not OBI is infectious by transfusion. All forms have been shown to be infectious in immunocompromised individuals, such as organ- or bone marrow-transplant recipients. In immunocompetent recipients, there is no evidence that anti-HBs-containing components (even at low titre) are infectious. Anti-HBc only, with HBV DNA, can be associated with infectivity, as can rare cases of HBV DNA without any serological HBV marker. If HBV nucleic acid amplification technology (NAT) is considered, the OBI viral load would usually be < 500 IU/ml, making testing of plasma pools unsuitable unless the sensitivity of NAT significantly increases by genome enrichment or test improvement.

Background and Objectives: Transmission of human parvovirus B19 (PV B19) by transfusion of blood and blood products is well documented. Although PV B19 infection is connected with severe complications in some recipients, donor screening is not yet mandatory. In this study the prevalence of PV B19, as detected by a haemagglutination assay (the Human PV B19 Antigen-Test), was assessed. In addition, the persistence of B19 DNA and the serological status of blood donors was also assessed. The specificity and utility of the Human PV B19 Antigen-Test for donor screening was investigated and compared with other screening strategies., Materials and Methods: The prevalence of PV B19 viraemia was assessed in 28 972 donations from 15,660 remunerated donors by means of the haemagglutination assay. Reactive results were confirmed by the polymerase chain reaction (PCR)., Results: Overall, 255 donations gave reactive or indeterminate results in the screening assay. Four donations/donors detected by the haemagglutination assay were confirmed as positive for B19 DNA by PCR. Therefore, a frequency was detected of 1:7243 B19-positive donations and 1:3915 positive donors. Specificity was determined to be 99.1%. Follow-up showed the persistence of viraemia in low concentrations for prolonged time-periods., Conclusion: Blood donations with a high level of human PV B19 viraemia can be detected by the haemagglutination assay, which is rapid and easy to perform. The presence of neutralizing antibody may inhibit specific haemagglutination.

Background and Objectives: West Africa is a highly endemic area for viral infections. The prevalence of five viral markers was determined in Ghanaian blood donors., Materials and Methods: Replacement and volunteer blood donors were screened using enzyme immunoassays (EIAs) for hepatitis B surface antigen (HBsAg), human immunodeficiency virus antibodies (anti-HIV), HIV p24 antigen, human T-cell lymphocytotrophic virus-I and -II antibodies (anti-HTLV-I/II) and hepatitis C virus antibodies (anti-HCV)., Results: HBsAg was present at an equally high frequency (15%) in young volunteer (median age 18 years) and older replacement (median age 33 years) blood donors. In contrast, the prevalence of anti-HIV and anti-HCV was significantly higher in replacement blood donors (2.4 and 0.3%, respectively, P < 0.001). HCV RNA was detected in 74 or 55% of seropositive donors, depending on the confirmatory criteria used. No p24 antigen-positive/anti-HIV-negative donations were found. The prevalence of HTLV-I/II was generally low (0.5%)., Conclusion: All blood donations should be screened for hepatitis B virus (HBV), HIV and HCV markers.

The increasing pace of development in molecular biological techniques during the last 10-15 years has had a direct effect on mass testing and diagnostic applications, including blood screening. Nucleic acid amplification techniques (NAT), usually based on the polymerase chain reaction (PCR), have been successfully applied to blood grouping and implemented recently in screening of blood donations for hepatitis C virus (HCV). The majority of microarray technologies involve an amplification step, yet the main benefits of this technology come from simultaneous analysis of thousands of analytes. Microarrays were developed to utilize the huge amount of information provided by genome projects, but they have clear potential in mass screening and diagnostics. The application of microarray technology may revolutionize blood testing, providing for the first time the prospect of an integrated platform for comprehensive donor and donation testing, replacing multiple individual assays. Design features of a blood-testing chip and various technologies with potential application in this field are discussed in this review.

Background and Objectives: Twenty-two laboratories from nine countries participated in an international collaborative study to establish a World Health Organization (WHO) international standard for hepatitis B virus (HBV) DNA nucleic acid amplification techniques (NAT)., Materials and Methods: Three samples, AA, BB (both of which were lyophilized) and CC (which was a liquid preparation), were analysed using several different NAT assays. The mean HBV DNA content of each sample was determined from the study., Results: Despite the range of assays (commercial and in-house) used by participants, there was good agreement among the overall mean 'equivalents'/ml obtained by the different assays, except for one laboratory (laboratory 4). The variation in estimates of log10 'equivalents'/ml was 1.75-1.25 for the three samples if results from laboratory 4 were excluded. The mean log10 'equivalents'/ml for all laboratories were 6.42 for sample AA, 6.30 for sample BB and 5.03 for sample CC (exclusion of results from laboratory 4 made little difference). The difference in titres between the two lyophilized samples (AA and BB) was not statistically significant but the titre of the frozen sample (CC) was significantly lower. Material AA (code 97/746) was accepted as the first WHO international standard for HBV DNA NAT assays and assigned a potency of 10(6) international units (IU)/ml., Conclusions: The titres (genome equivalents/ml) of three HBV preparations were determined by several laboratories using different NAT assays. This study enabled the establishment of an international standard, 97/746, for HBV DNA NAT assays.

Background and Objectives: Recent studies have suggested that potentially infectious donations provided during the antibody-negative 'window' phase of hepatitis C virus (HCV) infection may be identified by testing for viral RNA or HCV core protein. We therefore evaluated the performance of an HCV antigen enzyme-linked immunosorbent assay (ELISA) for identification of window-phase donations and for prospective screening of blood donors., Materials and Methods: One-hundred and twenty-eight archived plasma donations containing HCV RNA, but lacking antibody to HCV (anti-HCV), were tested by using the HCV antigen ELISA, together with 9951 freshly collected serum and plasma specimens from blood donors., Results: HCV core antigen was detected in 94% (120/128) of window-phase plasma donations. Overall specificity in freshly collected blood donor specimens was 99.74%. Two putative window-phase donations containing HCV core protein and viral RNA were identified from paid plasma donors by prospective testing with the HCV antigen ELISA., Conclusion: These results indicate that an HCV antigen ELISA can identify almost all (94%) of viraemic donations given during the seronegative window phase of infection. The performance of the HCV antigen ELISA appears to be suitable for large-scale screening of blood donations.

Background: We evaluated and analysed risk factors of HCV-infected blood donors according to HCV genotypes in order to improve the transfusion policy and safety of blood supply., Materials and Methods: HCV-RNA was analysed in sera from 518 anti-HCV-positive blood donors, who were invited to medical consultation and interview as to risk factors by means of an extensive questionnaire. HCV genotyping was done on all samples positive for HCV-RNA., Results: Of the 518 sera, 399 (77%) were HCV-RNA positive, and 394 of 399 HCV genotypes were identified. Major genotypes were 1b (34.3%), 3a (24%), 1a (19.5%) and 2 (11.4%). Of the donors, 289 (55.8%) were interviewed regarding their risk behaviour: 27% were former intravenous drug users (IVDUs), 26% had been transfused, 8% had a history of invasive diagnostic procedures, and 13% a history of surgery. Among the 224 interviewed donors, genotypes 1a and 3a were mainly associated with IVDU (51 and 45% respectively) and genotype 1b, with transfusion and nosocomial infections (40 and 25%, respectively)., Conclusion: In this population of anti-HCV-positive blood donors, nosocomial infection may be a route of HCV spread, but the main risk factor remains IVDU, particularly in young men. The transfusion policy will improve if predonation interviews of such young men are done with a specific and sensitive questionnaire., (Copyright 2000 S. Karger AG, Basel)

Background and Objectives: In January 1996, a case of hepatitis B virus (HBV) seroconversion in a multitransfused patient was reported to the blood bank From March through October 1995, the patient had received 23 units of red cells and 30 units of pooled platelet concentrates, encompassing an exposure to a total of 200 whole blood donations., Materials and Methods: In order to trace hepatitis B surface antigen (HBsAg)-negative but HBV-infectious blood donation(s), we tested samples of the donors obtained > or = 3 months after the implicated donations for anti-HBc (Corezyme EIA, Abbott). From 172/200 donors, archived samples of subsequent donations were available for this purpose. The remaining 28 donors were reinvited to the blood bank to obtain an additional blood sample for anti-HBc testing., Results: 1/200 follow-up donor samples was anti-HBc-positive. Retrospective testing of the implicated HBsAg-negative blood donation of this donor revealed anti-HBc-negative and HBV-DNA-positive results. The patient was transfused with the platelets of the HBV-infectious donation. On looking back, the other blood products prepared from this HBV-infectious donation caused posttransfusion HBV infection (PT-HBV) in 2 additional patients., Conclusion: Anti-HBc testing on mainly archived follow-up samples of 200 donors implicated in PT-HBV was a rapid, simple cost-effective and donor-friendly method to identify an infectious but HBsAg-negative, anti-HBc-negative and HBV-DNA PCR-positive blood donation. Routine anti-HBc screening would not have prevented this HBV transmission.

The detection of antibody to the second envelope protein (E2) of the hepatitis C virus (HCV) has been hampered by the lack of suitable antigens. A previously described E2 recombinant antigen (CHO-E2) expressed as a non-fused, highly glycosylated protein in mammalian cells was used to detect specific antibody (anti-E2) in samples from blood donors and viraemic patients showing positive or indeterminate results for anti-HCV after a wide serological study. Anti-E2 was detected in 50-75% of the donors positive for anti-HCV, 80% of viraemic immunocompetent patients with anti-NS3 alone and 28% of non-viraemic donors with anticore alone. In donors with anti-NS3 (15 samples) or anti-NS4 (51 samples) alone, anti-E2 was found occasionally (3 cases). Moreover, two anti-E2-positive samples from viraemic patients were misidentified by some commercial assays for screening anti-HCV. These results suggest that testing for anti-E2 may be useful for improving the performance of the current assays for anti-HCV screening and confirmation.

Seventy-three Swedish blood donors (52 men, 21 women; median age 36 years) repeatedly reactive for hepatitis C antibodies (anti-HCV C-100-3) were tested with a second-generation (2nd-gen) anti-HCV Elisa and a 4-band recombinant immunoblot assay (RIBA 2). These results were correlated to serum alanine aminotransferase (S-ALAT), liver morphology and viremia as detected by 'nested' polymerase chain reaction (PCR) based on primers from a 5'-noncoding sequence of the HCV genome. Thirty-five of 46 (76%) donors with positive 2nd-gen Elisa tests confirmed by RIBA 2 were PCR positive whereof 27 had histological findings compatible with chronic persistent hepatitis (CPH) and 7 had chronic active hepatitis (CAH). Ten of 56 (18%) 2nd-gen Elisa-positive donors were RIBA 2 negative (or indeterminate) and none of these had chronic hepatitis nor were PCR positive. Seventeen of 73 (23%) donors were 1st-gen Elisa positive but 2nd-gen Elisa negative. All of these were PCR negative and only 1 (6%) had chronic hepatitis (CPH). An elevated S-ALAT level (reference < 0.7 mu kat/l) was found in 26 2nd-gen Elisa and RIBA 2-positive donors of which 18 had CPH and 7 had CAH and all 25 were PCR positive. A normal S-ALAT level was found in 9 of 34 (26%) donors with chronic hepatitis (all had CPH) and positive PCR. We have found that blood donors with positive 2nd-gen anti-HCV Elisa tests confirmed by RIBA-2 and especially with a concomitant elevated S-ALAT are highly likely to be viremic as demonstrated by PCR and to have chronic hepatitis.